Limited Nosocomial Transmission of Drug-Resistant Tuberculosis, Moldova

Applying whole-genome-sequencing, we aimed to detect transmission events of multidrug-resistant/rifampin-resistant strains of Mycobacterium tuberculosis complex at a tuberculosis hospital in Chisinau, Moldova. We recorded ward, room, and bed information for each patient and monitored in-hospital transfers over 1 year. Detailed molecular and patient surveillance revealed only 2 nosocomial transmission events.

PP1 are admitted patients with new susceptible respiratory TB, to the PP2 -retreatment cases of susceptible respiratory TB, to the PP3 mainly patients with non-TB pulmonary disease and those with limited microbiological negative respiratory TB, to the MDRd -cases of MDR or rifampin resistant TB, and to the ETBS cases of extrapulmonary TB or those with pulmonary TB that require surgery treatment.
During the hospital admission the patients could be transferred between the wards. This usually occur when a patient admitted to a non-TB ward is diagnosed with TB or when in a TB patient a resistance pattern different of the initial pattern is documented. As well the transfer of the patients between the wards could occur if surgery or intensive care is required.

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During their stay in the hospital the patients are allowed to go outside for walk or smoking in the hospital yard where they interact with patients from other wards. As well, the patients share the elevator and use the same staircases. Also, the patients can come in contact in the hospital canteen, where are allowed only smear negative patients from PP1 and PP2. To mention that smear positive TB patient a not allowed to go to the canteen, they receive their food at a distribution point in their ward and consume it in their own rooms. All patients from MDRd and PP3 received the food exclusively at the distribution point in their department. Another possibility for a close contact between the patients from the same ward is the daily picking of the pills and injections at the nurse desk in the ward. Patients with susceptible TB receive a standard treatment with 4 drugs (isoniazid, rifampin, pyrazinamide, ethambutol) during the first 2-3 months (dependent of the achievement of the sputum culture conversion) followed by 4 months of isoniazid and rifampin. In most of the case the total treatment duration for susceptible TB is 6 months but could be prolonged in some cases.
During the study period, patients with MDR-TB initially started treatment with a standardized regimen of five 2 nd -line TB drugs including a fluroquinolone (levofloxacin or moxifloxacin), a 2 nd -line injectable (capreomycin or amikacin), ethionamide, cycloserine and pyrazinamide) which was adjusted, when necessary, once results of phenotypic or molecular drug susceptibility testing (DST) became available. The treatment duration in MDR TB patients is linked to the time of sputum culture conversion and consist of an initial 6-8 months of intensive phase followed by a continuation phase with a duration of 12-16 months.

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Usually in patients with smear positive susceptible TB, particularly in those with advanced disease, as well in MDR-TB patients treatment is initiated in TB hospital, where the patients are admitted at least until the smear sputum conversion is achieved and the final treatment scheme is decided. The treatment during hospital admission is supervised by a pneumologist specialized in treatment of TB patients (phthisio-pneumologist). After hospital discharge the patient continues to receive his treatment in outpatient conditions. In outpatient the treatment is provided by the patient's family doctor or phthisio-pneumologist who has his office closest to the patient domicile. The patient must come to the medical office daily to receive his medication under direct observation.

Microbiological methods
The standard battery of microbiological tests performed on admission for the detection of

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Phenotypic antimicrobial susceptibility testing MGIT cultures were incubated for a total of 6 weeks before being reported as negative.
The purity of the MGIT culture was checked by Ziehl-Neelsen stain, blood agar plate, and MPT64 antigen rapid test (BD Microbiology Systems, USA). For the preparation of the inoculation procedure, mycobacterial suspensions were used undiluted from day 1 to 2 following positivity, while the suspensions were diluted 1:5 with sterile saline from day 3 to 5.
Susceptibility testing using the BACTEC MGIT 960 system was performed with the following

Isolation of genomic DNA
M. tuberculosis complex strains were inoculated on Löwenstein Jensen medium at 37°C, until was good growth (abundant). Colonies were transferred to a microcentrifuge tube (2.0 ml) containing 400 μl TE buffer and heated for 20 min at 80°C to kill the bacteria. After 3 min centrifugation at 13,000 g we discarded the supernatant and added 400 μl TE-buffer, followed by vortexing to separate cells. We then added 50 μl lysozyme (10 mg/ml) vortexed briefly and incubated the solution overnight at 37°C. The next day, we added 70 μl 10% SDS, 5 μl proteinase K (10 mg/ml), vortexed softly and incubated the solution 10 min at 65°C.
Subsequently, we added 100 μl 5M NaCl, 100 μl CTAB/NaCl (pre-warmed at 65°C), followed by vortexing and incubation for 10 min at 65°C. we then added 750 μl Chloroform/Isoamyalcohol mix (24:1), inverted the tube few times and centrifuged at room Page 5 of 16 temperature for 15 min at 13,000 g. The aqueous supernatant was carefully transferred to a new microcentrifuge tube, and 0.6 volume isopropanol was added to precipitate the nucleic acids for 30 min at −20°C (or longer). We then centrifuged for 10 min at room temperature at 13,000 g, discarded the supernatant and washed the DNA in 0.5 ml of cold 75% Ethanol while inverting the tube few times, followed by 5 min centrifugation at room temperature and discarding the supernatant cautiously. The DNA-pellet was dried at 60°C for ≈10 min, and DNA was eventually dissolved in 100 μl TE-buffer at 37°C for 30 min or at room temperature until DNA was completely dissolved. Re-infection of the focus patients with an MDR/RR strain was confirmed by a pairwise genetic distance measurement exceeding 12 SNPs between the non-MDR/RR isolate and the Page 6 of 16 consecutive MDR/RR isolate. The MDR/RR isolate was further subjected to a transmission analysis to identify putative index cases.

Inference of patient-to-patient transmission
We used a maximum pairwise distance between any two isolates of 5 SNPs to identify molecular clusters as surrogate marker for recent transmission (4). A possible direct transmission between the admitted MDR/RR-TB patients and the focus patients were inferred when both isolates belonged to the same molecular cluster and shared identical genotypic drug resistance profiles. To visualize Mtbc isolates from patients who possibly acquired MDR/RR-TB in the hospital, we performed a core genome multi locus sequence type (cgMLST) analysis as described previously (5)

Statistical analysis
Continuous variables were expressed as the mean or median with standard deviation (SD) or inter quartile range (IQR) correspondingly, while categorical variables were presented as frequencies. For the comparisons of means and medians Student's t-test or Mann-Whitney test were applied. For the comparison of frequencies, the exact Fisher test was used. A p-value of <0.05 was considered significant.

General cohort characteristics
A total of 2,627 admissions were recorded at the Chiril Draganiuc Phthisiopneumology Institute during the study period. One hundred twenty-three patients had more than one Page 7 of 16 admission (109 patients had 2 admissions, 13 had 3 admissions and 1 had 4 admissions), amounting to 2,490 individual patient admissions during the study period. The mean age was 50.9 SD±17.4 years; 1623 (65.2%) were male. [is it of interest to add two sentences of differences between the wards?] Demographic and epidemiologic characteristics of the patients admitted to different hospital wards are presented in Table 1, main article.

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To point out putative transmission events between the focus patients and concurrently admitted MDR-TB patients, we performed a molecular cluster analysis based on pairwise genetic distance between all isolates (see methods). One patient (sample ID 24) was placed in the largest single cluster including 53 lineage 4.2.1 (URAL, cluster 26) isolates initially assuming an extensive ongoing nosocomial transmission scenario ( Figure 2). However, considering the low genetic diversity of the dominating lineage 4.2.1 (URAL) clade in the phylogeny (Figure 1), we have to assume that these strains are highly prevalent in the community in the Republic of Moldova. Furthermore, this patient (sample ID 24) did not have any identified hospital stay overlap with other MDR-TB patients in our cohort. Overall, 124/268 (46.3%) patients were part of one of the 28 identified clusters, including 7/17 focus patients (Appendix 2). Two patients (sample ID 28 and 37) were re-infected with the same pre-XDR strain (cluster 28) harboring identical resistance mediating mutations but lacking any hospital overlaps with each other or any other MDR-TB patient during their time on the non MDR-TB ward (Appendix 2). Likewise, another possible nosocomial re-infection event (cluster 27) involving a focus patient (sample ID 30) who was infected with the same strain (0 SNP difference) as another admitted MDR-TB patient was not plausible due to the absence of overlapping admission times (Appendix 2). In cluster 12 the focus patient (sample ID 12; MDR) overlapped for 4 days in the hospital with a pre-XDR patient, however, additional mutations conferring resistance against pyrazinamide and moxifloxacin in the putative index patient also excluded a direct link. Thus, only 2/17 focus patients (sample ID 14 and ID 285) had a possible direct link in the hospital with less than 6 SNP difference between the infecting Mtbc strains as well as 13 and 29 days overlap in the hospital with their putative index case (Table 2). For the other re-infection events, we need to assume a contact outside the hospital, an undetected mixed infection at baseline, or a missing culture from our cohort. on an MDR-TB ward from the beginning was similar 1-9 AFB in 100 fields (IQR 0 −1+ grade).
The baseline time to culture positivity, in MDR-TB patients admitted to the appropriate ward depended of the used media and was 15 days (IQR 12-20, n = 37) on MGIT and 55 days (IQR 52-55.5, n = 4) on LJ, while in those with initial admission to a non-appropriate ward was 16 days (IQR 11-23, n = 207) on MGIT and 38 days (29-50, n = 59) on LJ (Appendix 1 Table).
None of the differences were statistically significant.